Method for the selection of recombination clones comprising a sequence encoding an antidote protein to a toxic molecule

ABSTRACT

The present invention is related to a method for the selection of recombinant clones having integrated a gene of interest and a nucleotide sequence encoding a functional antidote protein to a toxic molecule, wherein said recombinant clones are the ones which survive following their integration into a host cell comprising in its genome a nucleotide sequence encoding said toxic molecule. The present invention is also related to a nucleic acid construct, a vector comprising said nucleic acid construct, a host cell and a cloning and/or sequencing kit for performing said method.

RELATED APPLICATIONS

This application is the U.S. National Phase of International ApplicationPCT/BE02/00021 filed on Feb. 22, 2002 designating the U.S. which waspublished in English as WO 02/066657 on Aug. 29, 2002, and which claimspriority to U.S. Provisional Application No. 60/271,204 filed Feb. 23,2001.

FIELD OF THE INVENTION

The present invention is in the field of the recombinant DNA technology.

More precisely, the present invention is related to a method, to anucleic acid construct, to a vector and to a cell used for the selectionof recombinant clones comprising a sequence encoding an antidote proteinto a toxic molecule.

BACKGROUND OF THE INVENTION AND STATE Of The Art

It is possible to clone DNA inserts into vectors without ever select forrecombinants and instead spend time in identification by hybridisationwith radio-labelled probes, screen by restriction of small-scalepreparations of plasmids or screening based on the inactivation of thealpha complementation in presence of X-Gal (blue/white screening).

These approaches used for more than one decade are not adapted to largescale cloning projects coming downstream complete genomes sequencingprograms. The sequence information available has increased tremendouslyand will increase further in the future.

In order to assess the biological function of a coding sequence whichhas been identified, its corresponding gene in the genome of an organismmust be specifically mutated (deleted or modified) (for instance in aknock-out mouse), which allows the study of phenotype(s) related to themutation introduced. The specificity of the mutation is given by atargeting vector (constructed in E. coli) containing homologousrecombination arms.

If the development of sequencing technologies has permitted to increasetremendously the number of sequenced genes, cloning and sub-cloninggenes at large scale in order to have recombinant clones and getinformation about the function of the corresponding genes is still alimiting step to functional genomic programs.

Cloning and sub-cloning each gene represent the bottleneck of the“functional genomic” programs. Therefore, new cloning approachesallowing speeding up these processes are required.

Because the identification of recombinants is a limiting step, it isclear that the positive selection of recombinants is required to movefrom traditional approaches to high throughput cloning allowing workingwith thousands of genes.

Cloning vectors permitting direct selection (positive selection) ofrecombinant strains have been proposed (for example, see Pierce et al.1992; Kuhn et al. 1986). However, most cloning vectors present thefollowing drawbacks: (i) they cannot be used to incorporate largenucleotide fragments, (ii) they are not easy to manipulate and (iv) theycannot be produced by a micro-organism in a large number of copieswithout bringing about the death of said micro-organism.

Furthermore, traditional restriction and ligase reactions can bereplaced by site-specific recombination and recombinants be selected bythe replacement of the ccdB gene (a member of the poison/antidote genefamily) by the insert of interest (U.S. Pat. Nos. 5,910,438, 6,180,407and International Patent WO 99/58652).

The main advantages of the CcdB-containing vectors over the otherpositive selection systems are i) the small size of their selective gene(ccdb: 303 bp), ii) the fact that the vector can be amplified in a hostharbouring a mutation that confers total resistance to the CcdB poison(gyrA462 resistant strain; Bernard and Couturier, 1992). Since E. coliis the host used for most molecular cloning strategies, it is importantto develop new systems which can enrich and widen the range of cloningpossibilities. The positive selection technology using CcdB has beenused to derive new vectors adapted to peculiar purposes: PCR cloningvectors (Gabant et al., 1997), vectors adapted for bacterial genetics(Gabant et al., 1998), and recently a kid gene belonging to the CcdBfamily has been used to design new cloning vectors (Gabant et al., 2000and WO01/46444).

Another example of the use of the CcdB gene is given by U.S. Pat. No.5,888,732. This document discloses the general principle of the cloningmethod known as “the Gateway system”. In this method, the traditionalrestriction and ligase reactions are replaced by site-specificrecombination sites and the recombinants are selected by inactivation(by deletion) of the ccdB gene by the gene of interest. This methodallows rapid and efficient transfer of all the genes from an organismfrom one vector to different vectors (i.e. expression vectors) byautomatic sub-cloning. The resulting sub-clones maintain the orientationas well as the reading frame allowing translation fusions (for a generaloverview, see Hartley et al., 2000).

However, said techniques are based upon counterselectable genesrequiring, the use of rpsl, tetR, sacB or ccdB counterselectable genes,which can generate a seamless second round product that carries no“scar” from the first round of recombination.

Counterselection (selection for the inactivation or deletion of a toxicgene) is typically less efficient than positive selection (acquisitionof a new property) as the intended recombination is only one of theseveral solutions for counterselection pressure. Any mutational eventthat ablates expression of the counterselectable genes, will also growunder counterselection pressure.

Thus for rare genetic events (which frequency can be compared to themutational inactivation of the counterselectable gene), candidates fromsecond-round counter-selection strategies, need to be screened to findthe intended recombination event.

In practice, it seems that the ratio of the intended to unwantedproducts varies widely (from <1% to 15%-85%) for reasons that are stillundefined (Muyrers G. P. P., Trends in Biochemical Sciences, Vol.26,no.5, p.325-331, 2001).

Aims Of The Invention

The present invention aims to provide a new and improved method andproducts which do not presents the drawbacks of the state of the art andwhich allow an improved positive selection of recombinant clones.

A specific aim of the present invention is to provide such method andproducts which allow the selection of rare genetic events, especiallythe selection of recombinant clones having integrated long DNAfragments.

Another aim of the present invention is to propose a method and productswhich are not affected or less affected by the development of resistancemutations in the cells used for said positive selection.

SUMMARY OF THE INVENTION

A first aspect of the present invention is related to a method for theselection of recombinant clones having integrated a gene of interest anda nucleotide sequence encoding a functional antidote protein to a toxicmolecule (to a cell, preferably a prokaryote cell), wherein saidrecombinant clones are the ones which survive following theirintegration into a host cell comprising in its genome a nucleotidesequence encoding said toxic molecule.

Another aspect of the present invention is related to products used toperform said method, especially to a nucleic acid construct comprisingat least one cassette nucleotide sequence made of at least onenucleotide sequence encoding an antidote protein to a toxic molecule (toa cell, preferably to a prokaryote cell) and a gene of interest or aninsertion site for said gene of interest (preferably, said insertionsite being not present in the nucleotide sequence encoding the antidoteprotein); said cassette nucleotide sequence being disposed between afirst recombination site and a second recombination site in said nucleicacid construct (said first recombination site and said secondrecombination site do not recombine with each other).

According to a preferred embodiment of the present invention, theantidote protein and the toxic molecule are respectively, an anti-poisonprotein and a poison protein. Said anti-poison or poison proteins couldbe wild type proteins or modified proteins which are naturally orartificially poisonous and affect one or more vital functions of a cell(preferably, a prokaryote cell) and may lead to the killing of the cell.

The antidote protein and the toxic molecule are preferably selected fromthe group consisting of CcdA/CcdB proteins, Kis/Kid proteins, Phd/Docproteins, SoK/HoK proteins, RelB/relE proteins, PasB (or PasC)/PasAproteins, mazF/mazE proteins or any other couple of anti-poison/poisonmolecules which are or are not of plasmid origin.

The toxic molecule can also be a toxin protein being naturally orartificially toxic and affecting one or more vital functions of a(prokaryote) cell.

The protein encoded by the gene sacB (from Bacillus amylolique-faciens),the protein GpE, the protein GATA-1 and the protein Crp are otherexamples of such toxic molecules.

The gene sacB encodes the levan sucrase which catalyses the hydrolysisof sucrose into products which are toxic for E. Coli (Pierce et al.Proc. Natl. Acad. Sci., Vol. 89, N°6 (1992) p. 2056-2060). The proteinGpE encodes the E genes from the bacteriophage φX174 which includes sixunique restriction sites and encodes gpE and which causes lysis of E.Coli cell (Heinrich et al., Gene, Vol. 42 n°3 (1986) p. 345-349). Theprotein GATA-1 has been described by Trudel et al. (Biotechniques 1996,Vol. 20(4), p. 684-693). the protein Crp has been described by Schlieperet al. (Anal. Biochem. 1998, Vol. 257(2), p. 203-209).

The antidote proteins to said toxic molecule are any protein able toreduce or suppress the effect of the corresponding toxic molecule on acell (preferably a prokaryotic cell), when said toxic molecule isproduced by said cell.

According to another embodiment of the present invention, the nucleotidesequence encoding the antidote protein is a first nucleotide fragmentencoding an inactive antidote protein, which can be rendered active(functional) following correct integration of the gene of interest inthe cassette sequence.

Preferably, the cassette sequence present in the nucleic acid constructaccording to the invention comprises also promoter/operator sequences inorder to obtain the expression of the antidote protein which can beexpressed constitutively or following the insertion of the gene ofinterest (insert) or by a recombination event.

For instance, the antidote activity can be achieved when the gene ofinterest (insert) brings a sequence comprising a transcriptional and/ortranslation signal that allows the expression of the antidote protein orwhen the gene of interest integrates another antidote sequence, aportion of it or its C-transcriptional or translation signal throughinsertion or by a recombination event.

By said mechanism, the correct insertion of the gene of interest insidethe nucleic acid construct can also be advantageously selected.

Said gene of interest may also further comprise a promoter/activatorsequence which allows or improves the expression of said antidoteprotein or comprises a second nucleotide fragment which will complementthe first nucleotide fragment of said antidote protein in order torender said antidote protein functional (active against the toxicmolecule).

According to a further embodiment of the present invention, theinsertion site of the gene of interest is a cloning site, such as arecombination site or a nucleotide sequence specifically cleaved by oneor more restriction enzymes.

Advantageously, the insertion site for said gene of interest iscomprised in a nucleotide sequence encoding a toxic molecule, preferablya poison protein. Therefore, the nucleic acid construct according to theinvention is based upon firstly, a negative selection which followingthe integration of the gene of interest into the nucleotide sequenceencoding the toxic molecule inactivates said sequence and secondly apositive selection which allows thereafter, the expression of anantidote protein.

This double selection allows, advantageously, an improved selection ofrecombinant clones with rare genetic events, such as the integration ofa very long DNA fragments, inside the nucleic acid construct accordingto the invention.

In the nucleic acid construct according to the invention, the first andthe second recombination sites, recognised by recombinase(s), arepreferably att phage λbased sites (specific recombination sites such asthe ones described by Ptashne M. et al. (Genetic switch, Cell Press,Cambridge, 1992). Preferably, said att sites are integrated into anucleic acid construct according to the invention by the methoddescribed by Landy A. et al. (Annual Review, Biochemistry, Vol.58,p.913, 1989)).

However, other types of recombination sites can be also integrated intothe nucleic acid construct according to the invention.

Another aspect of the present invention is related to a vector (anautonomously replicating vector such as a plasmid, a bacteriophage, avirus, a cationic vesicle or any other type of vector) comprising thenucleic acid construct according to the invention.

A preferred embodiment of the present invention is related to a vectordonor, DNA molecule comprising a first DNA segment and a second DNAsegment, said first or second DNA segment containing as selectablemarker at least one nucleotide sequence encoding an antidote protein toa toxic molecule, and wherein said first and second DNA segment isflanked by at least a first and second recombination site, which do notrecombine with each other.

Said selectable marker in the vector DNA molecule according to theinvention may also comprise at least one inactive fragment of theantidote sequence and a functional selectable marker is obtained, whenrecombining across said first and second recombination sites with afurther DNA segment comprising the inactive fragment of said selectablemarker (other fragment of the antidote sequence).

In the vector donor DNA molecule, the recombination sites are theabove-described recombination sites, preferably selected from the groupconsisting of the various recombination sites such as the ones describedin the U.S. Pat. No. 5,888,732.

The vector donor DNA molecule according to the invention isadvantageously combined in a kit of parts (preferably, in a cloning kit)with an insert donor DNA molecule, said insert donor DNA moleculecomprising the first DNA sequence flanked by the first recombinationsite and a second recombination site which do not recombine with eachother. The characteristics of said insert DNA molecules are the onesdescribed in the U.S. Pat. No. 5,888,732 and patents WO99/21977,WO01/31039, WO01/42509 incorporated herein by reference.

Another aspect of the present invention is related to a cell, possiblyintegrated in the above-mentioned kit, preferably a prokaryotic hostcell for the vector or the nucleic acid construct according to theinvention, said cell having incorporated in its chromosomal DNA, atleast one (preferably, at least two or more) nucleotide sequence(s)encoding the toxic molecule (for which the nucleotide sequence presentin the nucleic acid construct is encoding an antidote), the expressionof said toxic molecule being negatively repressed (controlled),preferably by a transcriptional and/or translation repressive tool, forinstance, by adding to said nucleotide sequence encoding the toxicmolecule a repressive promoter/operator nucleotide sequence which allowsa transcriptional and/or translation repressive control of said proteinpoison and avoid the death of the cell.

However, said repressive control can be removed by adding or suppressingthe addition of a specific compound to the cell (i.e.: a saccharide),Which allows the expression of the toxic molecule by the cell.

Furthermore, the activity of the toxic molecule could be alsoconditional (for example, by introducing in the cell a thermosensitiveallelle or a specific amber mutation suppression).

Furthermore, the cell could comprise also an antidote which is expressedin specific conditions and is able to block the deleterious effect ofsaid toxic molecule in the cell.

Preferably, the cell according to the invention is an E. coli cell whichhas the deposit number LMGP-21399.

Said cell was deposited in the Laboratorium voorMicrobiologie—Universiteit Généralement, K. L. ledeganckstraat 35,B-9000 Gent, Collection of the Belgian Coordinated Collection ofmicro-organisms, BCCM, under no. LMGP-21399, on the date of Feb. 15,2002). Said deposit has been made in accordance with the provision ofthe Budapest treaty regarding the international recommission of thedeposit of micro-organisms.

Another aspect of the present invention is related to the use of saidproducts for the selection of recombinants and to a method for selectingrecombinant clones comprising the step of providing a cassette sequencein the nucleic acid construct or the vector according to the inventionor inserting a gene of interest in the insertion site provided in saidcassette sequence, transform the cell according to the invention withthe nucleic acid construct or the vector according to the invention andselecting recombinant cell clones which survive.

The present invention will be described in more details in the followingnon-limiting example.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts a nucleic acid construct 1 according to the invention.

FIG. 2 is schematic representation of the CYS10 construct.

DETAILED DESCRIPTION OF THE INVENTION

Description of Plasmids and Strains

The FIG. 1 describes a nucleic acid construct 1 according to theinvention which comprises a cassette nucleotide sequence 2 made of anucleotide sequence 3, encoding an antidote protein 4 to a toxicmolecule 5 and a gene of interest 6 or an insertion site for a gene ofinterest 6, and a promoter/operator sequence 9; said cassette sequence 2being disposed between a first recombination site 7 and a secondrecombination site 8, said first and second recombination site(s) 7,8 donot recombine with each other.

Said nucleic acid construct 1 is advantageously integrated into a vectorwhich is an insert donor DNA vector 10 comprising a further selectablemarker 11. Said insert DNA donor vector 10 can be combined with aninsert acceptor DNA vector 12, comprising a recombination site 13 andanother selectable marker 14, said insert donor DNA vector 10 and saidinsert acceptor vector or molecule 12 after recombination form therecombinant vector 15 according to the invention. The correctrecombination orientation will be selected by using the characteristicsof the selective marker 11 and by using a bacterial strain expressing inits chromosome one or more genetic sequence(s) encoding the toxicmolecule 5.

In the present case, the nucleotide sequence encoding an antidoteprotein 4 is a nucleotide sequence encoding the CcdA protein, which isthe antidote to the toxic molecule 5 (CcdB protein), expressed by thebacteria.

The first selectable marker 11 presented in the insert donor DNA vector10 is the protein poison kid and the second selectable marker 14 is agenetic resistance to an antibiotic (ampicyline).

Advantageously, the insert donor DNA vector 10 could be amplified in abacteria which is resistant to the activity of the first selectablemarker 11, for instance, a bacteria expressing the antidote protein kisto the protein poison kid.

Such bacteria comprising constitutively the kis sequence is described inthe document WO01/46444 and protected by the deposit number LMGP-19171.

Another aspect of the present invention concerns the strain host cell 20wherein the selection of the recombinant clones is performed.

The preferred strain is an Escherichia coli strain CYS10, which is aderivative of DH10B strain (mcrA Δ(mrr-hsdRMS-mcrBC)Φ80lacZdM15 ΔlacW74endA1 recA1 deoR Δ(ara, leu)7697 araD139 ga/U ga/K nupG rpsL,commercialised by Invitrogen). This strain carries a ccdb poison gene 21under the control of the P_(tac) promoter 22 in the dcm gene of thechromosome, a defective λ expressing the Red and Gam functions under thecontrol of λ PERFLUOROCARBON LIQUID promoter and the temperaturesensitive CI857 repressor (descritpion of this system: Yu et al., 2000,PNAS 97:5978-5983) and two plasmids (Pulb3566 23 n Psc101Laclq 24). ThepULB3566 plasmid 23 produces the CcdA antidote 3 under the control ofthe P_(bad) promoter and carries the ampicilline resistance gene. ThePsc101 laclq 24 plasmid produces the Laclq repressor and carries thespectinomycine resistance gene. The Ptac promoter 22, controlling theexpression of the ccdB poison gene, is repressed by the Laclq protein,and is induced by the addition of IPTG 26 (isopropyl β-Dthiogalactoside, C₉H₁₈O₅S, Roche; 0,5 mmole/liter) in the culture media.However, even in the presence of the Laclq protein, the Ptac promoter 22is not completely repressed causing a residual expression. The Pbadpromoter, controlling the expression of the ccda antidote gene 3, iscompletely repressed in the absence of arabinose 25 and induced by theaddition of arabinose 25 (1%) in the culture media (Guzman et al., 1995,Journal of bacteriology, Vol.177, p.4121-4130).

Due to the temperature sensitive DI857 repressor, the strain grows onlyat 30° C. Due to the presence of ccdB in the chromosome, the straingrows only in the presence of arabinose allowing the production of theCcdA antidote. Consequently, the strain grows at 30° C., in LB mediumand in presence of arabinose (1%).

CcdA CcdB CYS10 strain Media production production at 30° C. LB +Ara(1%) Yes, induced Yes, residual Growth LB No, repressed Yes, residualNo growth LB + IPTG(0.5 mM) No, repressed Yes, induced No growth LB +Ara(1%) + Yes, induced Yes, induced Growth IPTG(0.5 mM)

The schematic representation of the CYS10 construction is presented inFIG. 2.

However, the characteristics of said bacterial strain can be improved bythe deletion of thermosensitive characteristic and by the introductionof additional genetic sequences encoding more toxic molecules in orderto reduce or avoid the selection of mutants which are resistant to theactivity of said toxic molecule (ccdB production).

The invention claimed is:
 1. A nucleic acid construct comprising acassette sequence disposed between a first recombination site and asecond recombination site, said cassette sequence comprising a firstnucleotide sequence encoding a first portion of an antidote protein to apoison protein wherein said first portion of said antidote protein isinactive and a site for the integration of a DNA insert, said antidoteprotein being reconstituted as functional antidote protein to the poisonprotein following insertion of a second nucleotide sequence encoding asecond portion of said antidote protein into the cassette, wherein theantidote and poison proteins are selected from the group consisting ofthe following coupled proteins: CcdA/CcdB protein, Kis/Kid protein,Phd/Doc protein, RelB/RelE protein, PasB/PasA protein, PasC/PasA proteinand mazE/mazF protein.
 2. The nucleic acid construct according to claim1, wherein the first recombination site and the second recombinationsite are att phage λ based sites.
 3. A vector which comprises thenucleic acid construct according to claim
 1. 4. The vector according toclaim 3, which is an autonomously replicating vector.
 5. A cloningand/or sequencing kit comprising the nucleic acid construct of claim 1or a vector which comprises said nucleic acid construct, said cloningand/or sequencing kit further comprising or a recombinant prokaryotichost cell having incorporated in its chromosomal DNA a recombinantnucleotide sequence encoding a poison protein, wherein the toxicity ofsaid poison protein can be overcome by a functional version of theantidote protein present in inactive form in said nucleic acid constructor vector and wherein said poison protein is selected from the groupconsisting of CcdB, Kid, Doc, RelE, PasA and MazF.
 6. The vector ofclaim 4, wherein said vector is a viral or a plasmid vector.
 7. Thenucleic acid construct according to claim 1, wherein the first andsecond recombination sites are recognized by a recombinase.
 8. The kitaccording to claim 5, wherein the first and second recombination sitesin said nucleic acid construct of claim 1 or said vector which comprisessaid nucleic acid construct are recognized by a recombinase.
 9. A vectorcomprising a cassette sequence comprising a first nucleotide sequenceencoding a first portion of an antidote protein to a poison proteinwherein said first portion of said antidote protein is inactive and asite for integrating a DNA insert, said antidote protein beingreconstituted as an functional antidote protein following integration ofthe DNA insert into the cassette sequence, wherein said DNA insertcomprises a second nucleotide sequence encoding a second portion of saidantidote protein and wherein the antidote and poison proteins areselected from the group consisting of the following coupled proteins:CcdA/CcdB protein, Kis/Kid protein, Phd/Doc protein, RelB/RelE protein,PasB/PasA protein, PasC/PasA protein and mazE/mazF protein.
 10. Thevector according to claim 9, which is an autonomously replicatingvector.
 11. A recombinant prokaryotic cell, which comprises the vectorof claim 9, and which has incorporated in its chromosomal DNA arecombinant nucleotide sequence encoding said poison protein.
 12. Thenucleic acid construct according to claim 1, wherein the nucleotidesequence comprising the insert comprises a promoter/operator sequence.13. A nucleic acid construct comprising a cassette sequence with a firstnucleotide sequence encoding a first portion of an antidote protein to apoison protein wherein said first portion of said antidote protein isinactive and a site for the insertion of a DNA insert, said antidoteprotein being reconstituted as a functional antidote protein to thepoison protein following integration of a second nucleotide sequenceencoding a second portion of said antidote protein into the cassettesequence, wherein the antidote and poison proteins are selected from thegroup consisting of the following coupled proteins: CcdA/CcdB protein,Kis/Kid protein, Phd/Doc protein, RelB/RelE protein, PasB/PasA protein,PasC/PasA protein and mazE/mazF protein.
 14. The nucleic acid constructof claim 1, wherein said DNA insert brings a sequence comprising atranscriptional and/or translational signal that allows the expressionof the antidote protein or wherein the DNA insert integrates anothernucleotide sequence encoding the antidote protein, a portion of theantidote protein or a C-terminal, transcriptional or translationalsignal of the antidote protein.
 15. The vector of claim 9, wherein saidDNA insert brings a sequence comprising a transcriptional and/ortranslational signal that allows the expression of the antidote proteinor wherein the DNA insert integrates another nucleotide sequenceencoding a C-terminal, transcriptional or translational signal of theantidote protein.
 16. The nucleic acid construct of claim 13, whereinsaid DNA insert brings a sequence comprising a transcriptional and/ortranslational signal that allows the expression of the antidote proteinor wherein the DNA insert integrates another nucleotide sequenceencoding a C-terminal, transcriptional or translational signal of theantidote protein.
 17. The recombinant prokaryotic cell of claim 11,wherein the transcription of said recombinant nucleotide sequenceencoding said poison protein is under the control of a repressiblepromoter.